JP2005110565A - Agent for keeping differentiation/pluripotency - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an agent for keeping differentiation/pluripotency, which keeps cell differentiation/pluripotency, especially an embryonic stem cell and somatic stem cell. <P>SOLUTION: The fusion protein comprises Nanog protein and TAT peptide. Since the agent for keeping differentiation/pluripotency reversibly adjusts the cell differentiation/pluripotency, the agent is clinically applied more than a case of a gene transfer method. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an agent for maintaining pluripotency of cells. Specifically, the present invention provides an agent that maintains the pluripotency of embryonic stem cells or somatic stem cells.

  Stem cells such as embryonic stem (ES) cells and somatic stem cells are expected as resources for transplantation therapy for neurological diseases, diabetes, leukemia and the like. ES cells are particularly valuable because they are derived from the inner cell mass of fertilized eggs (blastocysts) and have pluripotency. Mouse ES cells can maintain pluripotency by leukemia inhibitory factor (LIF). However, human and monkey ES cells are difficult to proliferate while maintaining full pluripotency even in the presence of LIF. An ES cell differentiation pluripotency maintenance substance that replaces LIF is desired. On the other hand, somatic stem cells do not use fertilized eggs, and thus have the advantage of not having ethical problems like ES cells. However, somatic stem cells do not proliferate better than ES cells, and it is difficult to obtain a sufficient number of cells.

The present inventor has identified a gene group ECAT (ES cell associated transcript) that is specifically expressed in ES cells and analyzed its function (Patent Document 1). So far, ECAT4 (Nanog) is a homeobox transcription factor, and it has been clarified that when it is overexpressed in ES cells, it can maintain pluripotency in a LIF-independent manner (Non-patent Document 1).
WO 02/097090 A1 Cell, 113, 631-642 (2003)

An object of the present invention is to provide a reversible differentiation pluripotency maintenance agent, particularly a reversible differentiation pluripotency maintenance agent for embryonic stem (ES) cells or somatic stem cells.
It is difficult to culture ES cells and somatic stem cells while maintaining pluripotency. Gene transfer of genes related to maintaining pluripotency can be achieved by known methods such as lipofection and retroviral vector methods, but (i) it is difficult to obtain high transfer efficiency, and (ii) the transferred gene is a chromosome. It is unclear where it is integrated into the chromosome, but it is irreversible and has a high risk of becoming cancerous. (Iii) If irreversibly maintaining pluripotency by gene transfer, then differentiate it and apply it Is difficult. Therefore, gene transfer methods are not realistic considering clinical applications.
Therefore, an object of the present invention is to provide a differentiation pluripotency maintenance agent that can be applied clinically without using gene transfer.

The present inventor has intensively studied to solve the above problems, and by adding a fusion protein in which a TAT peptide is combined with a Nanog (ECAT4) protein to the medium, the fusion protein is incorporated into RF8ES cells, and even without LIF. It was found that it proliferates while maintaining pluripotency. Furthermore, it was confirmed that when the fusion protein was removed from the medium, the cells differentiated. Since the protein taken up into the cell is degraded and the reaction is stopped, the maintenance of pluripotency of the cell according to the present invention is reversible.
As described above, the present invention can easily cultivate embryonic stem cells and somatic stem cells while maintaining the undifferentiated ability by incorporating the Nanog protein into the cells, and maintains the differentiation pluripotency of the cells. It has been completed based on the new knowledge that it can be reversibly adjusted.

That is, the gist of the present invention is as follows.
[1] A fusion protein comprising a protein transduction domain and a Nanog protein,
[2] The fusion protein according to [1] above, wherein the Nanog protein is derived from human, mouse or monkey,
[3] The fusion protein according to [1] or [2] above, wherein the protein transduction domain is HIV TAT, antenna pediatric homeodomain, HSV VP22 or a fragment thereof,
[4] The fusion protein according to [1] or [2] above, wherein the protein transduction domain is fibroblast growth factor (FGF), hepatocyte growth factor (HGF) or a fragment thereof,
[5] DNA containing a base sequence encoding the fusion protein according to any one of [1] to [4] above,
[6] An expression vector containing the DNA of [5] above,
[7] A cell containing the expression vector according to [6] above,
[8] The method for producing a fusion protein according to any one of [1] to [4] above, wherein the cell according to [7] is cultured under conditions capable of expressing the fusion protein,
[9] A chemical conjugate of Nanog protein and a compound having binding activity to a bisphosphonate compound, glucose-6-phosphate, or P-glycoprotein,
[10] A pluripotency maintenance agent containing the fusion protein according to any one of [1] to [4] or the chemical conjugate according to [9] as an active ingredient,
[11] The pluripotency maintenance agent according to [10] above, wherein the cell is an embryonic stem cell or a somatic stem cell,
[12] A method for maintaining pluripotency, comprising the step of contacting a cell with the fusion protein according to any one of [1] to [4] above or the conjugate according to [9] above,
[13] The method for maintaining pluripotency according to [12] above, wherein the cell is an embryonic stem cell or a somatic stem cell,
[14] The above [12] or [13], comprising the step of incorporating the fusion protein according to any one of [1] to [4] above or the chemical conjugate according to [9] above into cells by pinocytosis. The method for maintaining pluripotency as described,
[15] A cell containing the fusion protein according to any one of [1] to [4] or the chemical conjugate of [9] above.

  According to the present invention, the differentiation pluripotency of cells, particularly embryonic stem cells and somatic stem cells, can be easily maintained. The agent for maintaining pluripotency according to the present invention can be used for embryonic stem cells or somatic stem cells that are difficult to culture while maintaining pluripotency, and reversibly adjusts pluripotency. Therefore, clinical application is possible compared with the gene transfer method.

The “fusion protein” of the present invention is a fusion protein containing a protein transduction domain and a Nanog protein, and may be any one that maintains the differentiation pluripotency of a cell by being taken into the cell. By incorporating this fusion protein into cells, it becomes possible to culture cells, especially embryonic stem cells and somatic stem cells, while maintaining differentiation pluripotency, and improving the production efficiency and economy of these cells. Can be expected.
The fusion of the two domains of the fusion protein may be at any possible position, and the protein transduction domain may be fused to the N-terminus or C-terminus of the Nanog protein, or it may be fused to the active site of the Nanog protein. Preferably, the protein transduction domain is fused to the N-terminus of the Nanog protein.
The protein transduction domain may be fused to the Nanog protein by direct chemical bonding or via a linker molecule. In such a case, the linker molecule may be any divalent chemical structure capable of linking two domains. Preferred linker molecules of the invention are short peptides, such as those having 1-20 amino acid residues, preferably 1-10 amino acid residues.

As used herein, “pluripotency” means that ES cells proliferate in an undifferentiated state and can be differentiated into all cells including the reproductive system by transplanting to early mouse embryos. It can be differentiated into various cells even in vitro by simple induction.
As used herein, the term “protein” includes not only a protein represented by a specific amino acid sequence (SEQ ID NO: 2, 4 or 6), but also homologues thereof as long as their biological functions are equivalent. (Homologs and splice variants), mutants, derivatives and the like are included. Here, examples of homologs include proteins of other species such as mice and rats corresponding to human proteins, and these were identified by HomoloGene (https://www.ncbi.nlm.nih.gov/HomoloGene/) It can be identified a priori from the base sequence of the gene. Variants also include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletions, substitutions, additions and insertions. . Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein having no mutation.

  Accordingly, in the present specification, “Nanog protein” is used to encompass the Nanog protein represented by the specific amino acid sequence (SEQ ID NO: 2, 4 or 6), its homologues, mutants, derivatives and the like, unless otherwise specified. It is done. Specifically, mouse Nanog having the amino acid sequence set forth in SEQ ID NO: 2, human Nanog having the amino acid sequence set forth in SEQ ID NO: 4, monkey Nanog having the amino acid sequence set forth in SEQ ID NO: 6, and rat homolog Etc. are included. Here, the protein shown in SEQ ID NO: 2 is a protein encoded by a mouse-derived Nanog gene. The protein shown in SEQ ID NO: 4 is a protein encoded by a human-derived Nanog gene. The protein shown in SEQ ID NO: 6 is a protein encoded by the monkey-derived Nanog gene.

The Nanog protein includes not only a protein having each amino acid sequence shown in SEQ ID NO: 2, 4 or 6, but also homologues thereof. The homologue consists of an amino acid sequence in which one or more (usually several) amino acids are deleted, substituted or added in each of the amino acid sequences described above, and substantially the same as the Nanog protein of each original amino acid sequence. Mention may be made of proteins having the same quality of activity.
Here, the substantially homogeneous activity indicates a property that cells expressing the protein or cells incorporating the protein maintain differentiation pluripotency. Specifically, for example, mouse ES cells exhibit the property that the differentiation pluripotency of cells expressing the protein or cells incorporating the protein is maintained without LIF. Such properties of Nanog protein can be easily measured by a method known per se or a method analogous thereto. For example, chimeric mice can be made by transplanting cells into early mouse embryos, teratomas containing various cells can be formed when cells are transplanted into nude mice, or differentiation into cells can be induced by in vitro differentiation. The pluripotency can be measured by examining whether differentiation is possible.

The number of amino acid mutations and mutation sites in the protein are not limited as long as the activity is maintained. Indicators that determine how many amino acid residues need to be substituted, inserted or deleted without loss of activity are found using computer programs well known to those skilled in the art, such as DNA Star software be able to. For example, the number of mutations is typically within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids. The amino acid to be substituted is not particularly limited as long as the protein obtained after substitution retains the activity of Nanog protein. The amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution in terms of amino acid polarity, charge, solubility, hydrophobicity, hydrophilicity, amphiphilicity, etc. from the viewpoint of maintaining the structure of the protein. . For example, Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are amino acids classified as nonpolar amino acids, and Gly, Ser, Thr, Cys, Tyr, Asn and Gln are mutually uncharged amino acids. Asp and Glu are amino acids that are classified as acidic amino acids, and Lys, Arg, and His are amino acids that are classified as basic amino acids. Therefore, amino acids belonging to the same group can be appropriately selected using these as indices.
Nanog protein can be produced from a natural product (for example, ES cell line) by a known protein purification method, or by culturing a transformant containing a polynucleotide encoding Nanog protein described later. Can be manufactured.

In the present invention, the “protein introduction domain” is not particularly limited as long as it is capable of introducing a protein or assisting protein introduction. For example, the known HIV TAT, Antennapedia homeodomain, HSV VP22 or fragments thereof, cell penetrating peptides (CPP), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), Or the fragment etc. can be mentioned.
By using TAT, an antenna pediatric homeodomain, HSV VP22, or a fragment thereof as a protein transduction domain, ERas can be incorporated into a wide range of cells. In addition, tissue or cell-specific uptake can be achieved by using a substance that binds to a receptor specifically expressed in the target cell as a protein transduction domain, so that fibroblast growth factor (FGF), hepatocyte growth factor ( By using HGF) or a fragment thereof as a protein transduction domain, ERas can be incorporated into cells in a tissue-specific or cell-specific manner in which the corresponding receptor is expressed.

"HIV TAT, Antonnapedia homeodomain, HSV VP22" includes HIV-derived TAT (Green and Loewnstein, Cell, 56 (6): 1179-88 (1988); Frankel and Pabo, Cell, 55 (6): 1189-93 (1988)), Drosophila-derived antennapedia protein (Vives et al., J. Biol. Chem, 272 (25): 16010-7 (1997)), HSV-derived VP22 (Elliott and O'Hare, Cell, 88 (2): 223-33 (1997)), as well as their homologues (homologs and splice variants), mutants, etc., as long as their functions are equivalent. The Examples of mutants include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein without mutation.
"HIV TAT, antenna pediae homeodomain, fragment of HSV VP22" is any fragment of HIV TAT, antenna pediae homeodomain and HSV VP22 mentioned above, and assists protein introduction function or protein introduction What is necessary is just to have a function and the fusion protein can show activity. Preferably, a fragment of HIV TAT can be mentioned. The length of the fragment is not limited in any way as long as it has a function of assisting protein introduction or protein introduction. Protein transduction domains (hereinafter referred to as “PTDs”) having the ability to penetrate the cell membrane have been identified as fragments of HIV TAT, antenna pedia homeodomain and HSV VP22. A PTD is also included. It is known that it can be transduced into cultured cells by fusing a heterologous protein and PTD, and its production method is also known (Fawell et al., Proc. Natl. Acad. Sci. USA, 91). (2): 664-8 (1994); Elliott and O'Hare (1997), Phelan et al., Nature Biotech. 16: 440-443 (1998) and Dilber et al., Gene Ther., 6 (1) : 12-21 (1999), Patent No. 2702285). It has also been reported that β-galactosidase protein fused to 11 amino acid PTD derived from HIV TAT protein can invade all tissues of living mice and reach all single cells (Schwarze et al., Science , 285 (5433): 1569-72 (1999)).
Specific examples of the HIV TAT fragment include those described in the above-mentioned known literatures, preferably the HIV TAT fragment described in Patent No. 2702285, more preferably the sequence. It is the HIV TAT peptide shown by number: 11.

  “Fibroblast growth factor (FGF), hepatocyte growth factor (HGF) or a fragment thereof” is a known FGF protein, a known HGF protein or a fragment thereof, and the protein is introduced into the corresponding receptor-expressing cell. Any function may be used as long as it has a function of assisting protein introduction or a function to assist protein introduction. It is known that a conjugate with a fragment of fibroblast growth factor (FGF) is taken up into cells (Rojas, M. et al., Nat. Biotechnol., 16: 370-375 (1998), Lin, YZ et al., J. Biol. Chem. 270: 14255-14258 (1995)). Therefore, it is preferable to use the FGF fragment reported in them.

A DNA containing a base sequence encoding the fusion protein of the present invention contains a polynucleotide comprising a base sequence encoding the Nanog protein and a polynucleotide comprising a base sequence encoding a protein transduction domain.
The DNA containing the base sequence encoding the fusion protein of the present invention may be any of various cells and tissues, for example, ES cell-derived cDNA, genomic DNA, or synthetic DNA, or a combination thereof. Specifically, for example
(a) DNA containing a polynucleotide encoding the amino acid sequence described in SEQ ID NO: 2 and a polynucleotide encoding the amino acid sequence described in SEQ ID NO: 11;
(b) a DNA containing a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 4 and a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 11;
(c) DNA containing a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 6 and a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 11;
(d) DNA containing the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 9 or 10,
(e) DNA containing the base sequence set forth in SEQ ID NO: 3 and the base sequence set forth in SEQ ID NO: 9 or 10;
(f) a DNA comprising the base sequence set forth in SEQ ID NO: 5 and the base sequence set forth in SEQ ID NO: 9 or 10;
(g) a DNA comprising the nucleotide sequence represented by nucleotides 190 to 1104 of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 9 or 10,
(h) a DNA comprising the base sequence represented by the 217th nucleotide to the 1131st nucleotide of the base sequence described in SEQ ID NO: 3 and the base sequence described in SEQ ID NO: 9 or 10,
(i) a DNA comprising the base sequence represented by the 894th nucleotide to the 1691st nucleotide of the base sequence described in SEQ ID NO: 5 and the base sequence described in SEQ ID NO: 9 or 10,
(j) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(k) DNA containing the base sequence set forth in SEQ ID NO: 7;
(l) DNA consisting of the base sequence described in SEQ ID NO: 7,
Alternatively, DNAs containing substantially the same base sequence as those of (a) to (l) can be mentioned.

  These polynucleotides containing the nucleotide sequence described in SEQ ID NO: 1, 3 or 5 are hybridized with an appropriate part of the nucleotide sequence disclosed in SEQ ID NO: 1, 3 or 5 in the sequence listing of the present specification. For example, screening of an ES cell-derived cDNA library can be used. The cloning can be easily performed by those skilled in the art according to a basic book such as Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press (1989).

In addition, the DNA containing substantially the same base sequence as any of the DNAs of the above (a) to (l), specifically,
(m) DNA that hybridizes under stringent conditions to the complementary strand of the DNA of any one of (a) to (l) above,
(n) DNA containing a base sequence showing sequence identity with any of the DNAs of (a) to (l) above,
(o) a DNA encoding a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the protein encoded by the DNA of any one of (a) to (l),
Etc.

Here, the DNA that hybridizes under stringent conditions to the complementary strand of the DNA of any one of the above (a) to (l) is, for example, the base of the DNA of any of the above (a) to (l) About 40% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more of the sequence. Examples thereof include DNA containing the nucleotide sequence. Specifically, a partial sequence of the DNA of any one of (a) to (l) above can be mentioned.
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, a method described in a basic book such as Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press (1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.
“Stringent conditions” means, for example, hybridization at 42 ° C. in a solution containing 6 × SSC (20 × SSC represents 333 mM Sodium citrate, 333 mM NaCl), 0.5% SDS and 50% formamide. And conditions for hybridization at 65 ° C. in a solution containing 6 × SSC (not containing 50% formamide). The washing conditions after hybridization include washing conditions at 68 ° C. in a 0.1 × SSC, 0.5% SDS solution.

  The DNA containing a base sequence showing sequence identity with any of the DNAs of (a) to (l) is, for example, about 40% of the base sequence of the DNA of any of the above (a) to (l) Or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, still more preferably about 90% or more, most preferably about 95% or more. DNA to do. Specifically, a partial sequence of the DNA of any one of (a) to (l) above can be mentioned. DNA having such sequence identity can be prepared by the aforementioned hybridization reaction or PCR reaction, or by a DNA modification (deletion, addition, substitution) reaction described later.

DNA encoding a protein containing an amino acid sequence in which one or more amino acids have been deleted, substituted and / or added in the protein encoded by any of the DNAs of (a) to (l) above is artificially Means a DNA encoding a so-called modified protein or a protein such as an allelic variant present in the living body.
Here, the number of amino acid mutations and mutation sites in the protein are not limited as long as the activity of the protein encoded by the DNA of the present invention (the differentiation pluripotency maintenance activity) is retained. Indicators that determine how many amino acid residues need to be deleted, substituted and / or added without loss of activity in this way are computer programs well known to those skilled in the art, such as DNA Star. Can be found using software. For example, the number of mutations is typically within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids. In addition, from the viewpoint of maintaining the structure of the protein, the amino acid to be substituted may be an amino acid having properties similar to the amino acid before substitution, such as residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and amphiphilicity. preferable. For example, Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are amino acids classified as nonpolar amino acids, and Gly, Ser, Thr, Cys, Tyr, Asn and Gln are mutually uncharged amino acids. Asp and Glu are amino acids that are classified as acidic amino acids, and Lys, Arg, and His are amino acids that are classified as basic amino acids. Therefore, amino acids belonging to the same group can be appropriately selected using these as indices.

  The DNA encoding this modified protein can be produced by various methods described in basic books such as Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press (1989), such as site-directed mutagenesis and PCR. it can. Moreover, it can also manufacture in accordance with well-known methods, such as Gapped duplex method and Kunkel method, using a commercially available kit.

  As described above, the DNA of the present invention containing substantially the same base sequence as the DNA of any one of the above (a) to (l), the protein encoded by the DNA can be introduced into cells. It is possible that after introduction, a protein having substantially the same quality of activity as the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6 is preferable. Here, substantially the same quality of activity refers to the property that the differentiation pluripotency of a cell expressing a protein encoded by the DNA of the present invention or a cell incorporating the protein is maintained. Specifically, for example, mouse ES cells show the property that the pluripotency of cells expressing the protein or cells incorporating the protein is maintained without LIF. About this activity and its measuring method, it can implement by a well-known method.

  The DNA of the present invention can be in either single-stranded or double-stranded form. When the DNA of the present invention is double-stranded, a recombinant expression vector for expressing the protein of the present invention can be prepared by inserting the DNA of the present invention into an expression vector.

The expression vector used here can be appropriately selected according to the host to be used, the purpose, etc., and includes plasmids, phage vectors, virus vectors and the like.
For example, when the host is Escherichia coli, examples of the vector include plasmid vectors such as pUC118, pUC119, pBR322, and pCR3, and phage vectors such as λZAPII and λgt11. When the host is yeast, examples of the vector include pYES2, pYEUra3 and the like. When the host is an insect cell, pAcSGHisNT-A and the like can be mentioned. When the host is an animal cell, plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1, pRc / RSV, pRc / CMV, and viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors Is mentioned.

The vector may appropriately have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a selection marker gene, and a terminator.
Further, a sequence expressed as a fusion protein with thioredoxin, His tag, GST (glutathione S-transferase) or the like may be added so that isolation and purification can be facilitated. In this case, a GST fusion protein vector (such as pGEX4T) that has an appropriate promoter (such as lac, tac, trc, trp, CMV, or SV40 early promoter) that functions in the host cell, or a vector that has a tag sequence such as Myc or His. (PcDNA3.1 / Myc-His, etc.), and a vector (pET32a) that expresses a fusion protein with thioredoxin and His tag can be used.

By transforming the host with the expression vector prepared above, a transformed cell containing the expression vector can be prepared.
Examples of the host used here include Escherichia coli, yeast, insect cells, animal cells and the like. Examples of E. coli include HB101 strain, C600 strain, JM109 strain, DH5α strain, AD494 (DE3) strain and the like of E. coli K-12 strain. Examples of yeast include Saccharomyces cerevisiae. Examples of animal cells include L929 cells, BALB / c3T3 cells, C127 cells, CHO cells, COS cells, Vero cells, Hela cells, and 293-EBNA cells. Insect cells include sf9.

  As a method for introducing the expression vector into the host cell, a normal method suitable for the host cell may be used. Specific examples include a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a method using a lipid for gene transfer (Lipofectamine, Lipofectin; Gibco-BRL). After the introduction, a transformed cell in which the expression vector is introduced into the host cell can be selected by culturing in a normal medium containing a selection marker.

The protein of the present invention can be produced by continuing to culture the transformed cells obtained as described above under suitable conditions. The obtained protein can be further isolated and purified by general biochemical purification means. Examples of the purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography and the like. In addition, when the protein of the present invention is expressed as a fusion protein with the aforementioned thioredoxin, His tag, GST or the like, it can be isolated and purified by a purification method utilizing the properties of these fusion protein and tag.
As shown in the examples, these fusion proteins can be synthesized and purified in vitro.

The “chemical conjugate” of the present invention is a chemical compound obtained by chemically binding Nanog protein to a compound having binding activity to a bisphosphonate compound, glucose-6-phosphate, or P-glycoprotein. It is a conjugate.
Examples of the “bisphosphonate compound” include known compounds such as etidronate, alendronate, risedronate, incadronate, and pamidronate. Since bisphosphonate compounds are selectively taken up into osteoblasts, conjugates with bisphosphonate compounds using this known property have been reported (Calcif. Tissue Int., 59: 168-173). (1996), Bioorg. Med. Chem. Lett., 4: 1375-1380 (1995)). Therefore, a chemical conjugate in which a bisphosphonate compound is chemically bound to Nanog protein is specifically taken up by stromal stem cells that will be differentiated into osteoblasts in the future.

Glucose-6-phosphate is a known substance, and by chemically binding to Nanog protein, the chemical conjugate is specifically taken up by progenitor cells that will differentiate into hepatocytes and pancreatic cells in the future.
Examples of the compound having binding activity to P-glycoprotein include BCRP inhibitors. Specific examples include GF120918, which is a BCRP inhibitor. By chemically binding a compound having binding activity to P-glycoprotein and Nanog protein, the chemical conjugate is specifically taken up by various somatic stem cells called SP cells.

  Nanog protein can be made as described above. A purified Nanog protein and a chemical conjugate of these substances can be prepared by attaching them with a linker having an amino group and chemically binding them according to a known method such as acid amide bond.

The “pluripotency maintenance agent” of the present invention is an agent containing at least one of the fusion protein or the chemical conjugate of the present invention described above. An agent that maintains pluripotency. Specifically, for example, it refers to an agent that can be used for mouse ES cells so that the cells can maintain pluripotency even under conditions without LIF.
Here, the cells are not particularly limited, but may include mammalian cells. Mammalian cells are tissue / organ cells of mammals such as humans, monkeys, rats and mice, or cells derived therefrom, and may be any of individual cells, primary cells taken out from individuals, or cultured cells. Preferred examples include commercially available cultured cells (such as ATCC), stem cells such as embryonic stem (ES) cells and somatic stem cells, and more preferred are embryonic stem cells and somatic stem cells. The pluripotency maintenance agent of the present invention contributes to securing a sufficient number of cells in cell transplantation therapy by facilitating maintenance culture of embryonic stem cells and somatic stem cells, and facilitates the production of genetically modified animals. And The pluripotency maintenance agent of the present invention is also useful for increasing somatic stem cells in the body.

In order to cause the pluripotency maintenance agent of the present invention to act, an in vivo method in which the agent is directly introduced into the body, a certain type of cell is collected from a human, added to the cell outside the body, and the cell is incorporated into the body. There are ex vivo methods of returning and in vitro methods of adding to cultured cells.
As an administration method, if it is an ex vivo method or an in vitro method, it can be added to a culture medium in which cells are cultured or added directly to cells. The amount added can be appropriately adjusted depending on the cell type, the number of cells, etc., but it is sufficient that no cytotoxicity is observed and pluripotency maintenance activity is recognized. The addition amount of the fusion protein or conjugate of the present invention in the preparation is usually 0.0001 μM to 1000 μM, preferably 0.0001 μM to 10 μM, more preferably 0.0001 μM to 1 μM in a normal medium, and this is once a few days. It is preferable to add.
Examples of the administration method of the in vivo method include subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration and the like. The dose of the fusion protein or conjugate of the present invention in the preparation can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient, but is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 100 mg. Preferably it is 0.01 mg-10 mg, and it is preferable to administer this once to several days.

  The fusion protein or conjugate of the present invention, which is an active ingredient of a pluripotency maintenance agent, includes a pharmaceutically acceptable carrier (excipient, extender, binder, lubricant, etc.) as it is or as it is known per se. And can be prepared as a reagent or a pharmaceutical composition by mixing with conventional additives and the like, and may contain physiological saline, phosphate buffered saline (PBS), culture medium, or the like. The pharmaceutical composition can be adjusted to tablets, pills, capsules, powders, granules, syrups, injections, drops, external preparations, suppositories, and the like.

  The “differentiation pluripotency maintenance method” of the present invention is a method of maintaining differentiation pluripotency by contacting a cell with either the fusion protein or the chemical conjugate of the present invention. Specifically, it is a method of maintaining differentiation pluripotency by adding the differentiation pluripotency maintenance agent of this invention in a cell culture medium, making it contact with a cell, and making it take in a cell, for example. In the method for maintaining pluripotency of the present invention, either the fusion protein or the chemical conjugate of the present invention is brought into contact with a cell and then taken up by cell pinocytosis (phagocytosis). It may be a method in which sex is maintained. Uptake (introduction) using cell pinocytosis can be performed, for example, by using a commercially available kit, Influx (registered trademark) Pinocytic Cell-Loading Reagent (Molecular Probe).

Although a cell is not specifically limited, A mammalian cell can be mentioned. Mammalian cells are tissue / organ cells of mammals such as humans, monkeys, rats and mice, or cells derived therefrom, and may be any of individual cells, primary cells taken out from individuals, or cultured cells. Preferred examples include commercially available cultured cells (such as ATCC) and stem cells such as embryonic stem cells and somatic stem cells, and embryonic stem cells and somatic stem cells are more preferred.
Cells containing the fusion protein or chemical conjugate of the present invention can be produced by the differentiation pluripotency maintenance agent or the differentiation pluripotency maintenance method.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.

(1) Construction of TAT-Nanog expression vector Oligo DNA TAT-S (SEQ ID NO: 9) and TAT-AS (SEQ ID NO: 10) were denatured at 94 ° C for 1 minute and then gradually returned to room temperature to prepare double-stranded DNA. did. This was ligated to the HindIII / BamHI site of pCDNA3.1 (pCDNA3.1-TAT). Gateway rfB Casette was inserted into the BamHI / EcoRV site of this pCDNA3.1-TAT to prepare pCDNA3.1-TAT-GW. LR recombination reaction (Invitrogen) was performed with pCDNA3.1-TAT-GW and pEnter-mNanog to prepare pCDNA3.1-TAT-Nanog (FIG. 1). The DNA sequence of TAT-Nanog is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.
(2) Synthesis and purification of TAT-Nanog protein The fusion protein was synthesized and purified in vitro using the PureGene system.
(3) RF8ES cells derived from TAT-Nanog protein-maintained pluripotent mice remain undifferentiated for a long period of time when cultured in DMEM medium containing 15% fetal serum and 1000 U / μl LIF. When LIF is removed, the cells differentiate and turn into large, flat cells. When TAT-Nanog protein was added to the medium instead of LIF, the undifferentiated state was maintained without LIF (FIG. 2).

  According to the present invention, a fusion protein or chemical conjugate capable of maintaining the pluripotency of cells, particularly embryonic stem cells and somatic stem cells, can be provided. Since the differentiation pluripotency maintenance agent of this invention can adjust differentiation pluripotency reversibly, clinical application is possible compared with the gene transfer method.

Example of fusion protein An HIV-derived TAT peptide fused to the N-terminus of an ECAT protein such as Nanog or Nanog. These proteins can be synthesized and purified in E. coli or in the Cell Free synthesis system. Maintenance of undifferentiated state of ES cells by TAT-Nanog protein.

The base sequence shown in SEQ ID NO: 9 is oligo DNA TAT-S, the base sequence shown in SEQ ID NO: 10 is oligo DNA TAT-AS, and the amino acid sequence shown in SEQ ID NO: 11 is TAT peptide.

Claims (14)

A fusion protein comprising a protein transduction domain and a Nanog protein. The fusion protein according to claim 1, wherein the Nanog protein is derived from human, mouse or monkey. The fusion protein according to claim 1 or 2, wherein the protein transduction domain is HIV TAT, Antennapedia homeodomain, HSV VP22 or a fragment thereof. The fusion protein according to claim 1 or 2, wherein the protein transduction domain is fibroblast growth factor (FGF), hepatocyte growth factor (HGF) or a fragment thereof. DNA containing a base sequence encoding the fusion protein according to any one of claims 1 to 4. An expression vector containing the DNA according to claim 5. A cell containing the expression vector according to claim 6. The method for producing a fusion protein according to any one of claims 1 to 4, wherein the cell according to claim 7 is cultured under conditions capable of expressing the fusion protein. A chemical conjugate of Nanog protein and a compound having binding activity to bisphosphonate compound, glucose-6-phosphate, or P-glycoprotein. A pluripotency maintenance agent containing the fusion protein according to any one of claims 1 to 4 or the chemical conjugate according to claim 9 as an active ingredient. The pluripotency maintenance agent of Claim 10 whose cells are embryonic stem cells or somatic stem cells. A method for maintaining pluripotency, comprising the step of bringing the fusion protein according to any one of claims 1 to 4 or the chemical conjugate according to claim 9 into contact with a cell. The method for maintaining pluripotency according to claim 12, wherein the cells are embryonic stem cells or somatic stem cells. A cell containing the fusion protein according to any one of claims 1 to 4 or the chemical conjugate according to claim 9.

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